Fatty acid synthase (FAS) is over-expressed in many human cancers and its activity is required for cancer cell survival. To understand why FAS is over-expressed, we compared in breast cancer cells the utilization of fatty acids synthesized endogenously by FAS to those supplied exogenously in the culture medium. We found that endogenously synthesized fatty acids are esterified to the same lipid and phospholipid classes in the same proportions as those derived exogenously and that some endogenous fatty acids are excreted. Thus, FAS over-expression in cancer does not fulfill a specific requirement for endogenously synthesized fatty acids. We next investigated whether lipogenic activity mediated by FAS was, instead, involved in the maintenance of high glycolytic activity in cancer cells. By culturing breast cancer and non-cancer cells in anoxic conditions, we increased glycolysis 2-3 fold but observed no concomitant increase in lipogenesis. More research is needed to understand why FAS is over-expressed in cancer.

Šiame darbe siekta išsiaiškinti riebalų rūgščių vaidmenį reguliuojant oksidacinį fosforilinimą saponinu permeabilizuotose žiurkės širdies raumens skaidulose. Pagrindiniai darbo uždaviniai: 1).Įvertinti įvairios struktūros riebalų rūgščių vaidmenį reguliuojant oksidacinį fosforilinimą saponinu permeabilizuotose žiurkės širdies raumens skaidulose;2) Naudojant egzogeninę ADP-suvartojančią piruvato kinazės ir fosfoenolpiruvato sistemą įvertinti, ar oksiduojantis riebalų rūgštims kinta išorinės mitochondrijų membranos laidumas ADP-ui; 3) Tirti, ar riebalų rūgščių oksidacija veikia funkcinę sąveiką tarp kreatino kinazės ir ADP/ATP nešiklio; 4).Nustatyti riebalų rūgščių oksidacijos poveikį mitochondrijų in situ morfologijai bei įvertinti dekstrano T70 poveikį mitochondrijų in situ kvėpavimo parametrams ir morfologijai. Mitochondrijose in situ oksiduojantis įvairios struktūros riebalų rūgštims, vienoms ar mišinyje su piruvatu+malatu, oksidacinio fosforilinimo tariamoji KmADP sumažėja panašiu laipsniu lyginant su piruvato+malato oksidacija. Šis poveikis yra grįžtamas, t.y. riebalų rūgščių oksidacija nedaro įtakos po jos vykstančiai neriebalinės kilmės substratų oksidacijai. Oksiduojantis riebalų rūgštims išsaugoma funkcinė sąveika tarp kreatino kinazės ir ANT, nepaisant ženklaus tar. KmADP reikšmės sumažėjimo. Elektroninės mikroskopijos metodu įvertinome, kad riebalų rūgščių sąlygotas KmADP sumažėjimas gali būti susijęs su mitochondrijų struktūros pokyčiais, kuriuos sukelia riebalų... [to full text] / The aim of this study was to investigate the influence of fatty acid oxidation on the regulation of oxidative phosphorylation in permeabilized rat cardiac fibers. The objectives of the study:1). To evaluate the influence of different fatty acids in the regulation of oxidative phosphorylation in fibers; 2). To evaluate the changes in outer mitochondrial membrane permeability for ADP during fatty acid oxidation by the means of exogenous ADP consuming system consisting of pyruvate kinase and phosphoenolpyruvate;3); To investigate the effect of fatty acid oxidation on the functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase; 4). To investigate the effects of fatty acid oxidation and dextran T70 on the morphology and respiration of mitochondria in saponin-permeabilized rat cardiac fibers. The apparent Km of oxidative phosphorylation for ADP in saponin-permeabilized rat cardiac fibers is decreased several fold during oxidation of fatty acids alone or in the mixture with pyruvate compared to oxidation of pyruvate+malate. This effect is reversible, and fatty acid oxidation does not influence the subsequent oxidation of non-fatty substrates. The functional coupling between creatine kinase and adenine nucleotide translocase is not influenced by fatty acid oxidation and the efficiency of creatine kinase system does not depend on the nature of respiratory substrates. Analysis of electron microscopy images of fibres indicates that morphological... [to full text]

Feeding experiments were conducted on Atlantic pollock (Pollachius virens) to examine the variability in tissue fatty acid (FA) composition and stable carbon isotope fractionation of FA during digestion, assimilation and mobilization of lipids. The FA profiles and compound-specific carbon isotopes of chylomicrons, liver, muscle and fasted serum were compared to diet. FA analysis demonstrated similarity among tissue groups despite differences in feeding states. The FA results indicate the blood of post-prandial fish may serve as an alternative to tissue biopsies for the estimation of marine fish diets with compound-specific isotope analysis (CSIA). Despite similarity among FA profiles, the carbon isotope discrimination factors of FA varied independently, which suggests that fractionation is influenced by the degree to which individual FA are oxidized. These results provide preliminary information that is necessary in order to use CSIA to estimate the effects of fish diets.

Duplicated genes are present in the teleost fish lineage owing to a whole-genome duplication (WGD) event that occured ~ 230-400 million years ago. In the duplication-degeneration-complementation (DDC) model, partitioning of ancestral functions (subfunctionalization) and acquisition of novel functions (neofunctionalization) have been proposed as principal processes for the retention of duplicated genes in the genome. The DDC model was tested by analyzing the differential tissue-specific distribution of transcripts for the duplicated fatty acid-binding protein 10 (fabp10) genes in embryos, larvae and adult zebrafish (Danio rerio). The distribution of zebrafish fabp10a and fabp10b transcripts show a strikingly different tissue-specific pattern leading us to suggest that the zebrafish fabp10 duplicates had been retained in the genome owing to neofunctionalization. In another experiment to test the DDC model, transcriptional regulation of duplicated fabp genes was analyzed in zebrafish fed clofibrate, a peroxisome proliferator-activated receptor (PPAR) agonist. Clofibrate increased the steady-state level of both the duplicated copies of fabp1a/fabp1b.1, and fabp7a/fabp7b mRNA and heteronuclear RNA (hnRNA), but in different tissues of zebrafish. The steady-state level of fabp10a and fabp11a mRNA and hnRNA was elevated in liver of zebrafish, but not for fabp10b and fabp11b. We also investigated the effect of dietary fatty acids (FAs) and clofibrate on the transcriptional regulation of single copy fabp genes, fabp2, fabp3 and fabp6 in zebrafish. The steady-state level of fabp2 transcripts increased in intestine, while fabp3 mRNA increased in liver of zebrafish fed diets differing in FA content. In zebrafish fed clofibrate, fabp3 mRNA in intestine, and fabp6 mRNA in intestine and heart, was elevated. Whether the regulation of fabp gene transcription by clofibrate is controlled either directly or indirectly, the regulatory elements in the zebrafish fabp genes have diverged markedly since the WGD event, thereby supporting the DDC model.

Modifying milk fat composition to enhance its content of valuable fatty acids (FA) is required to meet the needs of a society which is becoming better informed about the relationship between diet and health. Manipulating the cow’s diet is an effective, natural way to modify the amount and composition of milk fat of cows. The two main factors that affect the cow’s diet concern management system (MS; pasture vs. confinement), and supplementation of diets with lipid supplements. Marine oils specifically are fed to enhance milk with n-3 long-chain polyunsaturated FA (n-3 LC-PUFA). The effects of source of marine lipid supplement (LS; fish oil vs. microalgae) in the cow’s diet and its interaction with MS on milk fat composition have not been studied. Thus, the main objective was to determine the interaction of MS and LS on milk FA profile and on expression of lipogenic genes in mammary, adipose and liver of lactating dairy cows. Compared with cows in confinement, grazing cows produced milk fat with lower content of unfavorable FA (12:0-16:0), while increasing the levels of beneficial FA including cis-9 18:1, 18:3 n-3 and conjugated 18:2. Feeding either fish oil or microalgae improved levels of n-3 LC-PUFA and reduced those of 16:0 in milk fat regardless of MS, but concurrently increased the level of other trans 18:1 isomers at the expense of trans-11 18:1. The reduced secretion of 12:0-16:0 in milk from grazing compared with confined cows was associated with lower mammary expression of lipogenic genes suggesting that part of the effect of MS on milk FA profile is mediated transcriptionally. The effect of LS on lipogenic gene expression was tissue specific with the greatest response to treatment observed in liver despite its minor role in lipogenesis in cattle relative to the mammary and adipose. Major conclusions were that milk produced in pasture systems has a more healthful FA profile than that of confinement systems, and that MS and LS have tissue specific effects on lipogenic gene expression in dairy cattle which have important effects on cow performance and healthfulness of the milk FA profile.

High linoleic acid soybeans facilitate maximum production of soy-based polyurethane. The objectives of this study were to: 1) Evaluate environmental influence on yield and seed composition traits; 2) Estimate correlation coefficients between linoleic acid with agronomic traits; 3) Validate SSR markers associated with fatty acid QTL in multiple environments and across diverse genotypes; and 4) Evaluate the influence of fertilizers differing in P and K concentrations on seed fatty acids. RG25 was identified as the best genotype to be commercialized for polyurethane production. Strong marker-trait associations across environments included Satt_335, Satt389, Satt556 associated with palmitic and stearic, Satt389 with oleic, Satt389 and Satt537 with linoleic acid. A significant increase in linoleic acid content was observed when plants received modified Hoagland’s solution with 2×K compared to without K. Development of a high linoleic acid soybean line for polyurethane production is feasible using validated SSR markers and high K fertility. / Ontario Ministry of Agriculture, Food and Rural Affairs

The biological activity of α-linolenic acid (ALA) is poorly understood and primarily associated with its conversion to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This study used the Δ6 desaturase knockout (D6KO) mouse, which lacks Δ6 desaturase and therefore cannot convert ALA, to evaluate the independent effects of ALA on preventing non-alcoholic fatty liver disease (NAFLD). First, the capacity of very long chain fatty acids to rescue the D6KO lipid profile was established. Next, to evaluate the independent effects of ALA, D6KO or wild-type mice were fed diets containing lard, canola, flaxseed, or fish oil. Following treatment, liver phospholipid fatty acid composition was evaluated and livers were scored for steatosis and inflammation. Glucose tolerance was also evaluated. D6KO mice fed ALA-rich diets had lower liver lipid accumulation, lower hepatic inflammation (8 weeks) and improved glucose tolerance (20 weeks) relative to lard-fed D6KO mice. Overall, this thesis supports an independent biological role for ALA. / D.W.L. Ma is funded by the Canola Council of Canada, Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation Leaders Opportunity Fund with matching from the Ontario Research Fund; Jessica Monteiro is funded Ontario Graduate Scholarship.

Fatty acid (FA) transport proteins are important regulators of FA uptake at the cell surface and the mitochondria where they are oxidized. Tight regulation of this process is necessary in order to meet metabolic requirements, while preventing excess lipid accumulation. In an obese state, there is an increase in FA uptake and increased storage of lipids in skeletal muscle, including diacylglycerol (DAG) and ceramides, which interfere with insulin-stimulated glucose uptake. Leptin administration has been shown to reduce muscle triacylglycerol accumulation and restore insulin response in obese rodents. However, it is not known whether this is mediated through a redistribution of the FA transport proteins to the cell surface and mitochondria. In addition to hyperglycemia, post-prandial lipidemia is also observed in the obese state, suggesting a resistance to insulin-stimulated FA uptake. The possibility that insulin-stimulated FA transporter translocation is impaired has received little attention. Lastly, while recent studies have demonstrated that the transverse (t)-tubules may be an important site for glucose uptake in muscle, this has not yet been examined with regards to the FA transporters. In the first study of this thesis, the recovery of insulin response with short-term (2 week) chronic leptin administration in high-fat fed rats was associated with a decrease in muscle reactive lipid species (DAG, ceramide) and an increase in markers of oxidative capacity. Contrary to our expectations, this was not mirrored by an alteration in the distribution of FA transport proteins (FAT/CD36 or FABPpm) at the sarcolemma or the two major mitochondrial populations. To gain further insight into FA transporters and their localization at the cell surface, the second study of this thesis analyzed both the sarcolemma and t-tubules (constitute 40 and 60% of the cell surface, respectively). The novel observation was made that the t-tubules contain FA transport proteins (FAT/CD36, FABPpm, FATP1 and FATP4), and that the distribution and response of these transporters to acute metabolic stimuli (insulin and muscle contraction) was unique from that of the sarcolemma. The third study of this thesis characterized the translocation of FA transport proteins in response to insulin in the obese, insulin resistant Zucker rat. FA transport proteins were chronically increased on both membrane fractions in muscle from the obese rats. Furthermore, a blunting of the insulin-induced translocation of FA transporters to both cell surface domains was observed, demonstrating that insulin resistance extends to the movement of FA as well as glucose transport proteins. The t-tubules appear to play an important role regarding substrate uptake. Together the data from this thesis suggests that a chronic elevation in FA transporters at both cell surface domains contributes to lipid accumulation in obese skeletal muscle, and that reduced sensitivity of both FA and glucose transport proteins to translocate in response to insulin may explain the lipidemia and hyperglycemia that often characterizes post-prandial situations in the obese condition. As the prevalence of obesity reaches epidemic proportions, research into the functional role of FA transport proteins in the progression of obesity related pathologies is warranted as we work to further our knowledge of this significant health issue. / Natural Sciences and Engineering Research Council, Canadian Institute of Health Research

This thesis is a series of investigations into the regulation of skeletal muscle mitochondrial metabolism. Novel regulatory mechanisms regarding mitochondrial fatty acid oxidation are continually being identified and alterations in skeletal muscle mitochondrial metabolism have been implicated in the pathogenesis of type II diabetes (T2DM). Therefore, advancing our basic understanding of mitochondrial regulatory processes is required to provide insight into the progression of T2DM. In study one, the utilization of knockout mice for the putative mitochondrial fatty acid transport protein FAT/CD36, showed that mitochondrial FAT/CD36 plays a functional role in mitochondrial long chain fatty acid (LCFA) oxidation. Specifically, FAT/CD36 was found to be located on the outer mitochondrial membrane (OMM) upstream of acyl-CoA synthetase. In study two, it was observed that in rat muscle, malonyl-CoA (M-CoA) inhibition kinetics of carnitine palmitoyltransferase I (CPT-I) display a more physiological IC50 in permeabilzed muscle fibre bundles (PmFB) compared to isolated mitochondria. These data suggest that the cytoskeleton may have a role in regulating M-CoA inhibition. Additionally, a significant effect of LCFA-CoA on M-CoA inhibition kinetics was observed. These data indicate that M-CoA content does not need to decrease to promote an increase in CPT-I flux. Finally, in a model of T2DM (ZDF rat), submaximal ADP-stimulated respiration rates and the content of adenine nucleotide translocase 2 (ANT2) content were depressed compared to lean control animals. Resveratrol treatment in ZDF rats recovered these declines concomitantly with improving insulin-stimulated skeletal muscle glucose uptake and the cellular redox state. A number of novel findings are presented, specifically, 1) a functional role for mitochondrial FAT/CD36 in mitochondrial LCFA oxidation was confirmed and the topology of this protein along the OMM is expanded upon, 2) M-CoA inhibition kinetics of CPT-I were re-evaluated in PmFB and a regulatory role of LCFA-CoA on M-CoA inhibition kinetics is established, and 3) submaximal ADP-stimulated respiration rates and ANT2 content are depressed in the ZDF rat and resveratrol supplementation prevents these decrements.

This thesis investigated the effects of omega-3 supplementation (2.0 g/day EPA + 1.0 g/day DHA) for 12 weeks on human skeletal muscle sarcolemmal and mitochondrial membrane fatty acid (FA) composition and whole body energy expenditure in young healthy males. Supplementation resulted in significant incorporation of EPA and DHA into sarcolemmal and mitochondrial membranes, with an increase in total unsaturation of mitochondrial membranes. The incorporation profile of the sarcolemma and mitochondria differed, with the mitochondria mimicking changes in whole muscle. There were no changes in the protein content of mitochondrial and selected proteins involved in energy metabolism, except for a significant increase in the long form of UCP3. Despite changes in membrane FA compositions, there were no changes in whole body substrate oxidation at rest or during exercise. These data demonstrate that omega-3 supplementation for 12 weeks altered the FA composition of sarcolemmal and mitochondrial membranes in human skeletal muscle.